The fixative is made by mixing 1 part of MES buffer (320 mM MES, 6.4 mM MgCl₂, 6.4 mM EDTA, 6.4 mM EGTA, pH 6.1), 2 parts of heptane and 1 part of 16% formaldehyde. The mixture will segregate into aqueous and organic phases. The specimens are dropped into the fixative and fixed for 15 min at room temperature with gentle rotation (not shaking or flipping) on a rotisserie-type mixer. Rotation ensures that the specimens pass through the interface regularly. Biphasic fixation preserves the microtubule structure relatively well and is mainly used for visualization of the microtubule. TNE buffer (10 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 7.4) is used in place of PBS in all subsequent staining procedures if visualizing microtubule is indeed the purpose.